Voltage-gated sodium ion channels (Navs) are integral to both neuronal and muscular signaling and thus are critical to life. The abnormal expression of Navs can lead to a series of ailments including epilepsy, arrythmia, intractable acute and chronic pain, metastatic cancers, erythermalgia, and congenital insensitivity to pain. Despite their importance to human disease, a detailed understanding of the dynamic functionality of these voltage-sensing transmembrane pores continues to elude the scientific community. In order to develop an understanding of how Navs sense and respond to voltage change, we will use molecular probes to construct a model of the tertiary structure of the Site II binding pocket within the ion conduction pore. While there are nine known binding sites distributed around the nine known isoforms of the mammalian Nav, only Site II is within the ion conduction pore of the protein. Within a series of lipophilic alkaloid natural products known to be selective Site II ligands, veratridine is the most efficacious agonist that is readily available from its natural source. Veratridine agonism of the Nav causes activation at normal resting potential, inhibition of inactivation, and reduction of bot single channel conductance and ion specificity. The behavior is extremely intriguing as Site II is located near the center of the conduction pore, however, veratridine ligation at that site profoundly effects the ion selectivity locus and the ion channel gate, both of which are located near the entrances to the pore many Angstroms away. A thorough explanation of how veratridine binding causes these changes will require an extremely detailed model of the veratridine binding pocket. In order to construct this model, ligand-protein complexes of veratridine and a series of related synthetic and semisynthetic molecular probes with a series of heterologously expressed point-mutated single isoform Navs will be characterized by whole-cell electrophysiology. These studies will be used to determine which functional groups on the natural product are essential for its biological activity as well as identify the residues within the ion pore of the Nav that ar integral for veratridine binding. The detailed understanding derived from these studies concerning how ligand binding at Site II deep within the ion pore of the Nav can have such a profound effect on the dynamic function of these transmembrane proteins could lead to the rational design of isoform-specific pharmaceuticals that would serve as treatment for a multitude of intractable diseases.